antibody storage - how to store? (Jul/15/2008 )

Hi all,I have a problem of antibody storageI purified my antibodies using Protein G column loading and washing buffer is 20mM Sodium phosphate buffer (pH 7.0)elution buffer is 100mM Glycine pH 2 and 3antibodies are neutralized in 100ul of 1M Tris pH 8.0 for 1ml fractions by collecting the eluates directly into tris containing tubesthe eluates with peak fractions were pooled and dialyzed against 20mM Tris pH 8.0, 40mM NaCl, 1mM EDTA and 20% glycerolafter dialysis, i aliquoted the antibodies and stored at -20 after snap freezing in liquid nitrogeni observed that there is loss of activity of antibody drasticallyafter this i tried storing the antibody without any freezing directly at 4Ceven after this the activity is lost within 15dayscan someone pease suggest me what is the thing i am doing wrong in the storage or even purification procedureawaiting for your valuable suggestionsLeelaram

-leelaram-

QUOTE (leelaram @ Jul 15 2008, 07:58 AM)

Hi all,I have a problem of antibody storageI purified my antibodies using Protein G column loading and washing buffer is 20mM Sodium phosphate buffer (pH 7.0)elution buffer is 100mM Glycine pH 2 and 3antibodies are neutralized in 100ul of 1M Tris pH 8.0 for 1ml fractions by collecting the eluates directly into tris containing tubesthe eluates with peak fractions were pooled and dialyzed against 20mM Tris pH 8.0, 40mM NaCl, 1mM EDTA and 20% glycerolafter dialysis, i aliquoted the antibodies and stored at -20 after snap freezing in liquid nitrogeni observed that there is loss of activity of antibody drasticallyafter this i tried storing the antibody without any freezing directly at 4Ceven after this the activity is lost within 15dayscan someone pease suggest me what is the thing i am doing wrong in the storage or even purification procedureawaiting for your valuable suggestionsLeelaram

Antibody storage:

Add Albumin &/or Glycerol if you plan to keep them at -20'C.Add azide if you plan to keep them at 4'C.

If you can add BSA as a bulking agent this may help. I would suspect the drastic pH change had an effect on the ab. Hopefully, after collection, you immediately adjusted the pH. I would dialyze against Hepes or other buffer that is used or would be used in tissue culture to mimick the environment the ab is commonly in.

Also, make sure you have a sufficient concentration 1 mg/ml would be ideal.

Azide or thimerosal for liquid storage.

Good luck.

-sgt4boston-

Hi,thank you for your valuable suggestionsi would try adding BSA but how much percentagealso will dialysis against PBS helpawaiting your repliesLeelaram

-leelaram-

If BSA will not interfere with the final use of your ab then 0.05% should be fine. I would strongly suggest HEPES for storage as it can be used for tissue culture.

-sgt4boston-

Hi all of you,thank you all for your valubale suggestionsI dialyzed my antibody with the procedure mentioned earlier in this topic against PBSand stored in 2% BSA. I observed that the antibody is pretty stable. and i stored in 4C.also i want to do some molecular biology experiments . hope this has solved the problemnow i want to transfer them to -20C. so can i place the vials directly into -20 or shall i have to snap freeze in liquid nitrogenawaiting your replies Leelaram

-leelaram-

QUOTE (leelaram @ Jul 24 2008, 03:19 PM)

Hi all of you,thank you all for your valubale suggestionsI dialyzed my antibody with the procedure mentioned earlier in this topic against PBSand stored in 2% BSA. I observed that the antibody is pretty stable. and i stored in 4C.also i want to do some molecular biology experiments . hope this has solved the problemnow i want to transfer them to -20C. so can i place the vials directly into -20 or shall i have to snap freeze in liquid nitrogenawaiting your replies Leelaram